In silico Homology Modeling and Epitope Prediction of NadA as a Potential Vaccine Candidate in Neisseria meningitidis

Neisseria meningitidis is a facultative pathogen bacterium which is well founded with a number of adhesion molecules to facilitate its colonization in human nasopharynx track. Neisseria meningitidis is a major cause of mortality from severe meningococcal disease and septicemia. Neisseria meningitidis adhesion, NadA, is a trimeric autotransporter adhesion molecule which is involved in cell adhesion, invasion, and antibody induction. It is identified in approximately 50% of N. meningitidis isolates, and is established as a vaccine candidate due to its antigenic effects. In the present study, we exploited bioinformatics tools to better understand and determine the 3D structure of NadA and its functional residues to select B cell epitopes, and provide information for elucidating the biological function and vaccine efficacy of NadA. Therefore, this study provided essential data to close gaps existing in biological areas. The most appropriate model of NadA was designed by SWISS MODEL software and important residues were determined using the subsequent epitope mapping procedures. Locations of important linear and conformational epitopes were determined and conserved residues were identified to broaden our knowledge of efficient vaccine design to reduce meningococcal infectioun in population. These data now provide a theme to design more broadly cross-protective antigens.

pathogen. It was reported that in special occasions the bacterium is able to move throughout the nasopharynx mucosal layer which can inevitably result in an invasive meningococcal disease including septicemia, and fulminate meningitidis (6,7). To induce disease, the bacterium needs a broad number of properties leading to colonization as well as invasion to the mucosal barriers, and eventually blood, resulting in septicemia followed by interaction with brain cell microvessels, and blood-brain barrier penetration (8)(9)(10). Adhesionassociated properties are due to type IV pili (Tfp), and the non-pilus adhesins which comprise opacity proteins Opa and Opc, and the auto transporter proteins App (adhesion penetration protein), NhhA (Neisseria hia homolog), and NadA (Neisseria adhesin A) (9,11).
There were numerous attempts to vaccine investigation. The first phases of efficient vaccines in adults but not in children were based on purified polysaccharides of four highly virulent serogroups of N. meningitidis (A, C, Y, and W-135). The next efforts were glycoconjugate-based vaccines against C serogroup which were highly successful in disease eradication caused by the mentioned serogroup in infants, children, and adults with 90% confidence. It is of paramount to approach surface exposed proteins in vaccine generation. These vaccines not only induce bactericidal antibodies, but may also eliminate further meningococcal diseases. Mentioned vaccines have been utilized in some countries but there are obstacles which should be tackled. The most important one is their weakness in supporting provision against heterologous strains due to the high sequence variability of protein antigens (12)(13)(14)(15).
In this study we focused on NadA, a potential virulence factor, an adhesion molecule, and vaccine candidate. NhhA and NadA are members of the oligomeric coiled-coil adhesion (Oca) family, also named trimeric auto transporters. NadA contributes to bacterial virulence due to its expression in near 50% of disease-associated isolates (8). More than 80% of isolates in hyper virulent lineages have nadA gene, and about half of the meningococcal isolates generate NadA (16,17). NadA antigen is also reported to be present in 52 out of 53 strains of hypervirulent lineage electerophoretic types (18).
Statistical association studies confirm this fact. In disparate geographical locations and time periods, isolates from the same clonal features have similar profiles for this antigen (19). Immunogenic nature of NadA is considerable, while it stimulates the immune system to produce high levels of bacterial antibodies and can be recognized by children's serum antibodies after invasive menangococcal disease (IMD) (20,21). The gene encoding NadA is associated with strains that belong to three of the four hypervirulent serogroup B (22,23). Lineages amino acids in C-terminal part have high similarity with that of the Yersinia adhesion YadA with special structure (17,24). The rate of nadA expression differs among isolates, and can be up to 100-fold. Moreover, it will be over expressed under in vivo condition due to the existence of special signals which are associated with the appropriate niche (25,26). NadA sequences are geneticaly divided into two categories with 45-50% amino acid identity. The first group consists of NadA1, NadA2, and NadA3 which are the most prevalent variants. It is exhibited that their sequence identity rate is 95%, and are cross-reactive from an immunogenic perspective. NadA4, NadA5, and NadA6 are members of the second group which are not common variants (24). In this report, we employed bioinformatics tools for 3D structure investigation regarding the fact that experimental determination is a considerable challenge since it is time-consuming, and its failure rate is relatively high (27). The first step in drug designing is in silico study to evaluate capability of candidate molecules. In silico studies can be used to determine the target structures for possible binding sites, identify candidate molecules, evaluate their capability to be considered as a drug, dock these molecules with the target, rank them according to their binding affinities, and refine structures to improve binding characteristics (28).
For outer membrane proteins such as NadA, purification and crystallization are cumbersome problems in addition to mentioned obstacles. Taken together, bioinformatics utilization can be more advantageous, and structure investigation is one of the wide applications of these tools in antigen determination and epitope mapping for designing new generation and highly effective vaccines.

Homologous structure search
To provide template structures the query protein sequence was used as an input data for the PSI-BLAST against protein data bank (PDB) at http://blast.ncbi.nlm.nih.gov/Blast.cgi.

General sequence characteristics
Protein sequence properties including molecular weight, theoretical pI, amino acid composition, total number of negatively and positively charged residues, instability index, and aliphatic index were obtained from an online software at http://expasy. org/tools/protparam. html, Protparam (30). su.se/ was also employed to determine membrane protein topology and signal peptides.

Secondary structure prediction
Self-optimized prediction method (SOPM) has been described to improve the efficiency of protein secondary structure prediction. The secondary structure of protein including helix, sheet, turn, and coil parameters was predicted at http://npsapbil.ibcp.fr/ cgibin/ npsa automat. pl? page = npsa sopma. html.

Ligand binding site prediction
Cofactor at http://zhanglab.ccmb.med.umich. edu/COFACTOR/ is a structure-based method for biological purpose notation of protein molecules.

Important amino acids involved in ligand binding
site are predicted by this server.

Functional and structural critical residues identification
Functional conserved residue allows more realism and robustness in the description of protein binding surfaces and epitope prediction. There are several ways to functionally and structurally annotate/predict important residues.
In this study, InterProSurf at http://curie.utmb. edu/pattest9.html was employed to predict functional sites on protein surface using patch analysis. NadA structure determined by previous strategies, served as an input file for this server.

Single-scale amino acid properties assay
Segments within NadA sequence that are likely to be antigenic were predicted using Bcepred at http://www.imtech.res.in/raghava/bcepred with accuracy of 58.7%. This server predicts B-cell epitopes using single of the physico-chemical properties (hydrophilicity, flexibility, mobility, accessibility, polarity, exposed surface and turns) or combination of them. Parameters such as hydrophilicity, flexibility, accessibility, turns and antigenic propensity of polypeptide have been correlated with the location of B cell epitopes.

Cleft and cavity analysis
Profunc at http://www.ebi.ac.uk/thornton-srv/ databases /profunc/ was used to predict clefts and grooves in the protein surface. Depth of clefts and amino acids located within the clefts are predictable by this software.  Figure 1.

Template search
The first illustrated result of PSI-BLAST against PDB data bank with 46% identity, 26% Alignment strategy has been utilized to manifest sequence similarities between the s elected target  are summarized in Table1.

Secondary structure and topology assessment
A 2D topology model of NadA was built based on predicted inside, transmembrane, and outside regions of the protein (Fig 2)    did not detect any such helix (Fig.3).

Homology modeling
Swiss model and phyre2 software were recruited for homology modeling. The former prepared 3 different models, and the latter introduced one model. All of them were selected for further analyses. The identity score was 46%.

Model evaluation
The best 3D model is shown in Figure 4 (Fig. 6a). Prosa revealed that the predicted model was among other acceptable proteins with z score = -4.75. (Fig. 6b).  Cofactor results illustrated cooperation of conserved residues in ligand binding site (189,192,194,196,197,198,200,201,202,203,209,210,216) with the highest C score LB which indicates the confidence of predicted binding site. Local similarity (sequence & structure) assessment between template binding site and predicted binding site in the query structure showed score 1.02 (BS-score>1) that represents a significant local match between the predicted and template binding site (Fig. 7).

Functional and structural critical residues
ConSurf and interproSurf annotated functional residues on the 3D structure of NadA. Results are shown in Figures 8a and b  Bold sequences indicate mentioned characteristics in protein sequence.

Single-scale amino acid properties assay
Properties such as hydrophilicity, accessibility, antigenicity, flexibility, and beta turn secondary structure in the protein sequence were predicted by Bcepred server ( "TSDDDVKKAA", "AEETKTN", "KADEAVKT-ANEAKQTAEETKQNV" residues are capable to be B cell epitopes.

Cleft analysis
ProFunc results showed a location with 12.17A depth, 57.64% accessible vertices, and 13.66% buried vertices that are the major cleft in NadA.

Prediction of B cell epitopes by integrated strategy
B-cell epitopes are regions on the surface of antigens that are recognized by B-cell receptors or specific antibodies. These epitopes can be categorized into two types: linear (continuous) and conformational (discontinuous) epitopes. Linear B cell epitopes were predicted by Bepipred server (Fig. 9). ABCpred result showed 9 hits of 16 mer peptide sequences as B-cell epitopes with ranking based on scores ( Table 3).

Discussion
There are many advantages for NadA to be an acceptable vaccine candidate. Relatively, NadA is involved in bacterial adhesion due to the surface exposure, and is expressed in 50% of virulent strains and all three hypervirulent lineages. It also antibodies (19). According to the NadA location and its chemical characteristics (Table 1)